We are studying the regulation and function of the p53 tumor suppressor protein. Although p53 is not necesary for normal growth and development, it is an essential component of the stress response that functions to protect cells from the acquisition of potentially tumorigenic changes. Activation of p53 in response to signals such as DNA damage or oncogene activation leads to either cell cycle arrest or apoptotic cell death. p53 activity is lost in almost all human cancers, either by mutation within the p53 gene or through defects in the pathways that normally allow activation of the p53 response. We are therefore hoping that a clearer understanding of the p53 pathway will allow us to reactivate the p53 response in cancer cells, and so target these cells for apoptotic cell death. Many stress signals can activate the p53 response, and we have shown that multiple pathways exist to transduce these signals. The induction of p53 by deregulated cell growth that occurs after oncogene activation, or following loss of the pRB tumor suppressor pathway, is mediated, at least in part, by the p14ARF protein. We and others have shown that p14ARF functions by binding to MDM2, the principal regulator of p53 stability in cells. MDM2 functions as a ubiquitin ligase for both p53 and itself, and we have shown that this activity depends on the RING finger motif in MDM2. p14ARF can inhibit MDM2 E3 activity, and also leads to the relocalization of MDM2 from the nucleoplasm to the nucleolus. This relocalization appears to contribute to the activation of p53 by physically separating p53 (which remains in the nucleoplasm) from MDM2, and we showed that nucleolar sequestration of MDM2 depends on nucleolar localization signals within MDM2 itself. It appears that p14ARF binding reveals these sequences in MDM2, so allowing relocalization of MDM2, and we are now pursuing the possibility that other modifications of MDM2, such as phosphorylation, might have the same effect. Finally, we have shown that a small peptide from p14ARF can function like the wild-type protein in activation of p53, providing a good target for the development of small molecules that could induce p53 activity in some tumor cells. One of the rationales for reactivation of p53 as a tumor therapy is that cancer cells show an enhanced sensitivity to p53-mediated apoptosis compared to many normal cell types. We have recently shown that a second transcription factor, NF-kB, is an essential component of the apoptotic response to p53, although NF-kB is not required for p53-induced cell cycle arrest. These results are particularly important in light of previous studies showing that NF-kB can also function to protect cells from some other apoptotic signals, revealing opposing activities of NF-kB that depend on which apoptotic pathway is activated. Clinical trials using NF-kB inhibitors are underway to try and sensitize tumor cells to chemotherapy. While this approach is likely to be effective in tumor cells with mutant p53, inhibition of NF-kB may be counterproductive in those tumors that retain wild-type p53, since our studies suggest that this would decrease p53-mediated apoptosis in these cells.